Methods for regulating gastrointestinal motility

ABSTRACT

Methods for reducing gastric motility and delaying gastric emptying for therapeutic and diagnostic purposes are disclosed which comprise administration of an effective amount of an exendin or an exendin agonist. Methods for treating conditions associated with elevated, inappropriate, or undesired post-prandial blood glucose levels are disclosed which comprise administration of an effective amount of an exendin or an exendin agonist alone or in conjunction with other anti-gastric emptying agents.

RELATED APPLICATION

This application is continuation-in-part of U.S. patent application Ser.No. 08/694,954 filed Aug. 8, 1996 now abandoned, the contents of whichare hereby incorporated by this reference.

FIELD OF THE INVENTION

The present invention relates to methods for regulating gastrointestinalmotility. More particularly, the invention relates to the use ofexendins and analogs and agonists thereof for the treatment of disorderswhich would be benefited with agents useful in delaying and/or slowinggastric emptying.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to that invention.

Publications and other materials including patents and patentapplications used to illuminate the specification are herebyincorporated by reference.

Exendin

The exendins are peptides that are found in the venom of theGila-monster, a lizard found in Arizona. Exendin-3 [SEQ. ID. NO. 1] ispresent in the venom of Heloderma horridum, and exendin-4 [SEQ. ID. NO.2] is present in the venom of Heloderma suspectum (Eng, J., et al., J.Biol. Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem.,267:7402-05, 1992). The exendins have some sequence similarity: toseveral members of the glucagon-like peptide family, with the highesthomology, 53%, being to GLP-1 [7-36]NH₂ (Goke, et al., J. Biol. Chem.,268:19650-55, 1993). GLP-1 [7-36]NH₂ [SEQ. ID. NO. 3] is also known asproglucagon [78-107], or simply the shorthand “GLP-1,” which is usedinterchangeably with GLP-1 [7-36]NH₂ throughout this application. Thesequences of exendin-3, exendin-4 and GLP-1 are shown in FIG. 1. GLP-1has an insulinotropic effect, stimulating insulin secretion frompancreatic β-cells; GLP-1 also inhibits glucagon secretion frompancreatic α-cells (Ørskov, et al., Diabetes, 42:658-61, 1993;D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). GLP-1 is reportedto inhibit gastric emptying (Willms B, et al., J. Clin Endocrinol Metab81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci 38 (4): 665-73,1993), and gastric acid secretion. Schjoldager B T, et al., Dig Dis Sci34 (5): 703-8, 1989; O'Halloran D J, et al., J Endocrinol 126 (1):169-73, 1990; Wettergren A, et al., Dig Dis sci 38 (4): 665-73, 1993).GLP-1 [7-37], [SEQ. ID. NO. 37] which has an additional glycine residueat its carboxy terminus, also stimulates insulin secretion in humans(Ørskov, et al., Diabetes, 42:658-61, 1993).

A transmembrane G-protein adenylate-cyclase-coupled receptor believed tobe responsible for the insulinotropic effect of GLP-1 has been clonedfrom a β-cell line (Thorens, Proc. Natl. Acad. Sci. USA 89:8641-45(1992), hereinafter referred to as the “cloned GLP-1 receptor.”Exendin-4 is reportedly a potent agonist at GLP-1 receptors oninsulin-secreting βTC1 cells, at dispersed acinar cells from guinea pigpancreas, and at parietal cells from stomach; the peptide is alsoreported to stimulate somatostatin release and inhibit gastrin releasein isolated stomachs (Goke, et al., J. Biol. Chem. 268:19650-55, 1993;Schepp, et al., Eur. J. Pharmacol, 69:183-91, 1994; Eissele, et al.,Life Sci., 55:629-34, 1994). Exendin-3 and exendin-4 were found to beGLP-1 agonists in stimulating cAMP production in, and amylase releasefrom, pancreatic acinar cells (Malhotra, R., et al., RegulatoryPeptides, 41:149-56, 1992; Raufman, et al., J. Biol. Chem. 267:21432-37,1992; Singh, et al., Regul. Pept. 53:47-59, 1994). Based on theinsulinotropic activities of exendin-3 and exendin-4, their use has beenproposed for the treatment of diabetes mellitus and the prevention ofhyperglycemia (Eng, U.S. Pat. No. 5,424,286).

In contrast to the full-length exendins, truncated exendin peptides suchas exendin [9-39], [SEQ. ID. NO. 4] a carboxyamidated molecule, andfragments 3-39 through 9-39 of exendin have been reported to be potentand selective antagonists of GLP-1 (Goke, et al., J. Biol. Chem.,268:19650-55, 1993; Schepp, W., et al., Eur. J. Pharm. 269:183-91, 1994;Montrose-Rafizadeh, et al., Diabetes,45(Suppl. 2):152A, 1996). Exendin[9-39], the sequence of which is shown in FIG. 1, reportedly blocksendogenous GLP-1 in vivo, resulting in reduced insulin secretion. Wang,et al., J. Clin. Invest., 95:417-21, 1995; D'Alessio, et al., J. Clin.Invest., 97:133-38, 1996). Exendins and exendin [9-391] bind to thecloned GLP-1 receptor (Fehmann H C, et al., Peptides 15 (3): 453-6,1994; Thorens B, et al., Diabetes 42 (11): 1678-82, 1993). In cellstransfected with the cloned GLP-1 receptor, exendin-4 is an agonist,i.e., it increases cAMP, while exendin [9-39] is an antagonist, i.e., itblocks the stimulatory actions of exendin-4 and GLP-1.

Exendin [9-39] is also reported to act as an antagonist of the fulllength exendins, inhibiting stimulation of pancreatic acinar cells byexendin 3 and exendin 4 (Raufman, et al., J. Biol. Chem. 266:2897-902,1991; Raufman, et al., J. Biol. Chem., 266:21432-37, 1992). Exendin[9-39] is said to inhibit the stimulation of plasma insulin levels byexendin 4, and inhibits the somatostatin release-stimulating and gastrinrelease-inhibiting activities of exendin-4 and GLP-1 (Kolligs, F., etal., Diabetes, 44:16-19, 1995; Eissele, et al., Life Sciences,55:629-34, 1994).

Agents which serve to delay gastric emptying have found a place inmedicine as diagnostic aids in gastro-intestinal radiologicexaminations. For example, glucagon is a polypeptide hormone which isproduced by the a cells of the pancreatic islets of Langerhans. It is ahyperglycaemic agent which mobilizes glucose by activating hepaticglycogenolysis. It can to a lesser extent stimulate the secretion ofpancreatic insulin. Glucagon is used in the treatment of insulin-inducedhypoglycaemia when administration of glucose intravenously is notpossible. However, as glucagon reduces the motility of thegastro-intestinal tract it is also used as a diagnostic aid ingastro-intestinal radiological examinations. Glucagon has also been usedin several studies to treat various painful gastro-intestinal disordersassociated with spasm. Daniel, et al. (Br. Med. J., 1974, 3, 720)reported quicker symptomatic relief of acute diverticulitis in patientstreated with glucagon compared with those who had been treated withanalgesics or antispasmodics. A review by Glauser, et al., (J. Am. Coll.Emergency Physns, 8:228, 1979) described relief of acute oesophagealfood obstruction following glucagon therapy. In another study glucagonsignificantly relieved pain and tenderness in 21 patients with biliarytract disease compared with 22 patients treated with placebo (M. J.Stower, et al., Br. J. Surg, 69:591-2, 1982).

Methods for regulating gastrointestinal motility using amylin agonistsare described in International Application No. PCT/US94/10225, publishedMar. 16, 1995.

SUMMARY OF THE INVENTION

The present invention concerns the surprising discovery that exendinsare potent inhibitors of gastric emptying. Exendins and exendin agonistsare useful as inhibitors of gastric emptying for the treatment of, forexample, diabetes mellitus, obesity, the ingestion of toxins, or fordiagnostic purposes.

The present invention is directed to novel methods for reducing gastricmotility and slowing gastric emptying, comprising the administration ofan exendin, for example, exendin 3 [SEQ ID NO. 1], exendin 4 [SEQ ID NO.2], or other compounds which effectively bind to the receptor at whichexendins exert their action on gastric motility and gastric emptying.These methods will be useful in the treatment of, for example,post-prandial hyperglycemia, a complication associated with type 1(insulin dependent) and type 2 (non-insulin dependent) diabetesmellitus.

In a first aspect, the invention features a method of beneficiallyregulating gastrointestinal motility in a subject by administering tosaid subject a therapeutically effective amount of an exendin or anexendin agonist. By “exendin agonist” is meant a compound which mimicsthe effects of exendins on gastric motility and gastric emptying,namely, a compound which effectively binds to the receptor at whichexendins exert their action on gastric motility and gastric emptying,preferably an analog or derivative of an exendin.

Exendin agonist compounds useful in present invention include thosecompounds of the formula (I)

1                  5                      10 Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄Xaa₅ Xaa₆ Xaa₇ Xaa₈                  15                  20 Ser Lys GlnXaa₉ Glu Glu Glu Ala Val Arg Leu                25                    30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu LysAsn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Zwherein Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ isAsp or Glu₄; Xaa is Phe, Tyr or naphthalanine; Xaa₅ is Thr or Ser; Xaa₆is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu, Ile, Val, pentylglycineor Met; Xaa₉ is Leu, Ile, pentylglycine, Val or Met; Xaa₁₀ is Phe, Tyror naphthalanine; Xaa₁₁ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr,or naphthylalanine; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either SEQ. ID. NOS. 1 or 2. Also useful in the presentinvention are pharmaceutically acceptable salts of the compounds offormula (I).

In one embodiment, the methods of the present invention are directed toreducing gastric motility. In another embodiment, the invention isdirected to methods of delaying gastric emptying.

These methods may be used on a subject undergoing a gastrointestinaldiagnostic procedure, for example radiological examination or magneticresonance imaging. Alternatively, these methods may be used to reducegastric motility in a subject suffering from a gastrointestinaldisorder, for example, spasm (which may be associated with acutediverticulitis, a disorder of the biliary tract or a disorder of theSphincter of Oddi).

In another aspect, the invention is directed to a method of treatingpost-prandial dumping syndrome in a subject by administering to thesubject a therapeutically effective amount of an exendin or exendinagonist.

In yet another aspect, the invention is directed to a method of treatingpost-prandial hyperglycemia by administering to a subject atherapeutically effective amount of an exendin or exendin agonist. In apreferred embodiment, the post-prandial hyperglycemia is a consequenceof Type 2 diabetes mellitus. In other preferred embodiments, thepost-prandial hyperglycemia is a consequence of Type 1 diabetes mellitusor impaired glucose tolerance.

In another aspect, a therapeutically effective amount of an amylinagonist is also administered to the subject. In a preferred aspect, theamylin agonist is an amylin or an amylin agonist analog such as^(25,28,29)Pro-human-amylin. The use of amylin agonists to treatpost-prandial hyperglycemia, as well as to beneficially regulategastrointestinal motility, is described in International Application No.PCT/US94/10225, published Mar. 16, 1995 which has been incorporated byreference herein.

In yet another aspect, a therapeutically effective amount of an insulinor insulin analog is also administered, separately or together with anexendin or exendin agonist, to the subject.

In another aspect, the invention is directed to a method of treatingingestion of a toxin by administering an amount of an exendin or anexendin agonist effective to prevent or reduce passage of stomachcontents to the intestines and aspirating the stomach contents.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise.

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers iftheir structure allow such stereoisomeric forms. Natural amino acidsinclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited toazetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,N-methylglycine, N-methylisoleucine, N-methylpentylglycine,N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,pentylglycine, pipecolic acid and thioproline. Amino acid analogsinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or modified on their N-terminalamino group or their side-chain groups, as for example, methioninesulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone.

The term “amino acid analog” refers to an amino acid wherein either theC-terminal carboxy group, the N-terminal amino group or side-chainfunctional group has been chemically codified to another functionalgroup. For example, aspartic acid-(beta-methyl ester) is an amino acidanalog of aspartic acid; N-ethylglycine is an amino acid analog ofglycine; or alanine carboxamide is an amino acid analog of alanine.

The term “amino acid residue, refers to radicals having the structure:(1) —C(O)—R—NH—, wherein R typically is —CH(R′)—, wherein R′ is an aminoacid side chain, typically H or a carbon containing substitutent; or(2), wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid,proline or pipecolic acid residues, respectively.

The term “lower” referred to herein in connection with organic radicalssuch as alkyl groups defines such groups with up to and including about6, preferably up to and including 4 and advantageously one or two carbonatoms. Such groups may be straight chain or branched chain.

“Pharmaceutically acceptable salt” includes salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid. In practice the use of the salt formamounts to use of the base form. The compounds of the present inventionare useful in both free base and salt form, with both forms beingconsidered as being within the scope of the present invention.

In addition, the following abbreviations stand for the following:

-   -   “ACN” or “CH₃CN” refers to acetonitrile.    -   “Boc”, “tBoc” or “Tboc” refers to t-butoxy carbonyl.    -   “DCC” refers to N,N′-dicyclohexylcarbodiimide.    -   “Fmoc” refers to fluorenylmethoxycarbonyl.    -   “HBTU” refers to        2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium        hexaflurophosphate.    -   “HOBt” refers to 1-hydroxybenzotriazole monohydrate.    -   “homoP” or hPro” refers to homoproline.    -   “MeAla” or “Nme” refers to N-methylalanine.    -   “naph” rekgers to naphthylalanine.    -   “pG” or pGly refers to pentylglycine.    -   “tBuG” refers to tertiary-butylglycine.    -   “ThioP” or tpro” refers to thioproline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the amino acid sequences of GLP-1,exendin-3, exendin4 and exendin [9-39] using standard single letterrather than three letter amino acid codes.

FIG. 2 shows GLP-1 [7-36]NH₂, exendin-3 and exendin-4 dose-responseeffects of prior subcutaneous injection on the retention of gastriccontents 20 minutes after gavage in normal rats (n=3-17 for each point).Symbols are means±SEM and the curves define the best fitting logisticfunctions. “Zero” indicates the fraction of gastric contents retained inuntreated normal rats.

FIG. 3 shows the dose response effects of prior injection of exendin-4(n=29), exendin-4 acid (n=36) and ¹⁴Leu,²⁵Phe exendin-4 (n=36) on theretention of gastric contents 20 minutes after gavage in normal rats.Symbols are means plus or minus standard error of the mean and thecurves define the best fitting logistic functions. “Zero” indicates thefraction of gastric contents retained in untreated normal rats.

FIG. 4 shows the effect of prior injection of 1.0 μg exendin-4 (sc),n=6; 1.0 μg exendin-4 (sc) plus 0.3 mg exendin [9-39] (sc), n=6; and 0.3mg exendin [9-39] (sc), n=6 on the retention of gastric contents 20minutes after gavage. Also shown are saline controls at t=0 and t=20min. The error bars show standard error of the mean. As shown in FIG. 4,exendin-4 alone potently inhibited gastric emptying. Exendin [9-39] (sc)alone had no effect on gastric emptying. When injected along withexendin-4, exendin [9-39] did not antagonize the effect of exendin-4 ongastric emptying inhibition.

FIG. 5 shows the effect of prior injection of 0.3 μg exendin-4 (sc), n=5and 0.3 μg exendin-4 (sc) plus 0.5 mg exendin [9-39] (iv), n=5 on theretention of gastric contents 20 minutes after gavage. Also shown aresaline controls at t+0 and t=20 min. The error bars show standard errorof the mean. As shown in FIG. 5, exendin-4 alone potently inhibitedgastric emptying. When injected along with exendin-4, exendin [9-39](iv) did not antagonize the effect of exendin-4 on gastric emptyinginhibition.

FIG. 6 shows the effect of prior injection of 10 μg GLP-1 [7-36]NH₂(sc), n=8; 10 μg GLP-1 [7-36]NH₂ (sc) plus 3 mg exendin [9-39] (sc),n=6; and 0.3 mg exendin [9-39] (sc), n=6 on the retention of gastriccontents 20 minutes after gavage. Also shown are saline controls at t=0and t=20 min. The error bars show standard error of the mean. As shownin FIG. 6, GLP-1 [7-36]NH₂ potently inhibited gastric emptying. Exendin[9-39] (sc) alone had no effect on gastric emptying. When injected alongwith GLP-1 [7-36]NH₂, exendin [9-39] did not antagonize the effect ofGLP-1 [7-36]NH₂ on gastric emptying inhibition.

FIG. 7 shows the effect of prior injection of 10 μg GLP-1 [7-36]NH₂(sc), n=8, and 10 μg GLP-1 [7-36]NH₂ (sc) plus 0.5 mg exendin [9-39](iv), n=3 on the retention of gastric contents 20 minutes after gavage.Also shown are saline controls at t=0 and t=20 min. The error bars showstandard error of the mean. As shown in FIG. 7, GLP-1 [7-36]NH2 alonepotently inhibited gastric emptying. When injected along with GLP-1[7-36]NH₂, exendin [9-39] (iv) did not antagonize the effect of GLP-1[7-36]NH₂ on gastric emptying inhibition.

FIGS. 8A and 8B depict the amino acid sequences for certain exendinagonists [SEQ. ID. NOS. 5 TO 35].

DETAILED DESCRIPTION OF THE INVENTION

Exendins and exendin agonists (including exendin analogs and exendinderivatives) are useful in this invention in view of theirpharmacological properties. Activity as exendin agonists can beindicated by activity in the assays described below. Effects of exendinsor exendin agonists on gastric motility and gastric emptying can beidentified, evaluated, or screened for, using the methods described inExamples 1-3 below, or other art-known or equivalent methods fordetermining gastric motility. Negative receptor assays or screens forexendin agonist compounds or candidate exendin agonist compounds, suchas a GLP-1 receptor preparation, an amylin receptor assay/screen usingan amylin receptor preparation as described in U.S. Pat. No. 5,264,372,issued Nov. 23, 1993, the contents of which are incorporated herein byreference, one or more calcitonin receptor assays/screens using, forexample, T47D and MCF7 breast carcinoma cells, which contain calciumreceptors coupled to the stimulation of adenyl cyclase activity, and/ora CGRP receptor assay/screen using, for example, SK-N-MC cells, can beused to evaluate and/or confirm exendin agonist activity.

One such method for use in identifying or evaluating the ability of acompound to slow gastric motility, comprises: (a) bringing together atest sample and a test system, said test sample comprising one or moretest compounds, said test system comprising a system for evaluatinggastric motility, said system being characterized in that it exhibits,for example, elevated plasma glucose in response to the introduction tosaid system of glucose or a meal; and, (b) determining the presence oramount of a rise in plasma glucose in said system. Positive and/ornegative controls may be used as well.

Exendins and exendin agonist compounds such as exendin analogs andexendin derivatives, described herein may be prepared through peptidepurification as described in, for example, Eng, et al., J. Biol. Chem.265:20259-62, 1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992,hereby incorporated by reference herein. Alternatively, exendins andexendin agonist peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al. J.Biol. Chem. 267;21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer. Typically,an α-N-carbamoyl protected amino acid and an amino acid attached to thegrowing peptide chain on a resin are coupled at room temperature in aninert solvent such as dimethylformamide, N-methylpyrrolidinone ormethylene chloride in the presence of coupling agents such asdicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of abase such as diisopropylethylamine. The α-N-carbamoyl protecting groupis removed from the resulting peptide-resin using a reagent such astrifluoroacetic acid or piperidine, and the coupling reaction repeatedwith the next desired N-protected amino acid to be added to the peptidechain. Suitable N-protecting groups are well known in the art, witht-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) beingpreferred herein.

The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.). The side-chain protected amino acids, suchas Boc-Arg(Mts), Fmoc-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu),Boc-Ser(Bzl) Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu),Boc-Lys(Cl-Z), Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu),Fmoc-His(Trt), Fmoc-Asn(Trt), and Fmoc-Gln(Trt) may be purchased fromApplied Biosystems, Inc. Boc-His(BOM) may be purchased from AppliedBiosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole,methylsulfide, phenol, ethanedithiol, and thioanisole may be obtainedfrom Aldrich Chemical Company (Milwaukee, Wis.). Air Products andChemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic acid andmethanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).

Solid phase peptide synthesis may be carried out with an automaticpeptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (option 1) system and tBoc or Fmoc chemistry(see, Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resinsmay be cleaved with HF (−5° C. to 0° C., 1 hour). The peptide may beextracted from the resin with alternating water and acetic acid, and thefiltrates lyophilized. The Fmoc-peptide resins may be cleaved accordingto standard methods (Introduction to Cleavage Techniques, AppliedBiosystems, Inc., 1990, pp. 6-12). Peptides may be also assembled usingan Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).Peptides may be purified by RP-HPLC (preparative and analytical) using aWaters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ,2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, andpurity may be determined using a C4, C8 or C18 analytical column (5μ,0.46×25 cm; Vydac) Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) maybe delivered to the analytical column at a flowrate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. The peptides may be hydrolyzed by vapor-phase acid hydrolysis(115° C., 20-24 h). Hydrolysates may be derivatized and analyzed bystandard methods (Cohen, S. A., Meys, M., and Tarrin, T. L. (1989), ThePico Tag Method: A Manual of Advanced Techniques for Amino AcidAnalysis, pp. 11-52, millipore Corporation, Milford, Mass.). Fast atombombardment analysis may be carried out by M-Scan, Incorporated (WestChester, Pa.). Mass calibration may be performed using cesium iodide orcesium iodide/glycerol. Plasma desorption ionization analysis using timeof flight detection may be carried out on an Applied Biosystems Bio-Ion20 mass spectrometer.

Peptide compounds useful in the invention may also be prepared usingrecombinant DNA techniques, using methods now known in the art. See,e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor (1989). Alternatively, such compounds may be preparedby homogeneous phase peptide synthesis methods.

The use of exendin analogs or derivatives is included within the methodsof the present invention. Exendin analogs or derivatives are functionalvariants having similar amino acid sequence and retaining, to someextent, at least the gastric motility- and gastric emptying-relatedactivities of the related exendin. By “functional variant” is meant ananalog or derivative which has an activity that can be substituted forone or more activities of a particular exendin. Preferred functionalvariants retain all of the activities of a particular exendin, however,the functional variant may have an activity that, when measuredquantitatively, is stronger or weaker, as measured in exendin functionalassays, for example, such as those disclosed herein. Preferredfunctional variants have activities that are within about 1% to about10,000% of the activity of the related exendin, more preferably betweenabout 10% to about 1000%, and more preferably within about 50% to about500%. Derivatives have at least about 15% sequence similarity,preferably about 70%, more preferably about 90%, and even morepreferably about 95% sequence similarity to the related exendin.“Sequence similarity” refers to “homology” observed between amino acidsequences in two different polypeptides, irrespective of polypeptideorigin.

The ability of the analog or derivative to retain some activity can bemeasured using techniques described herein.

Derivatives include modification occurring during or after translation,for example, by phosphorylation, glycosylation, crosslinking, acylation,proteolytic cleavage, linkage to an antibody molecule, membrane moleculeor other ligand (see Ferguson et al., Annu. Rev. Biochem. 57:285-320,1988).

Specific types of analogs include amino acid alterations such asdeletions, substitutions, additions, and amino acid modifications. A“deletion” refers to the absence of one or more amino acid residue(s) inthe related polypeptide. An “addition” refers to the presence of one ormore amino acid residue(s) in the related polypeptide. Additions anddeletions to a polypeptide may be at the amino terminus, the carboxyterminus, and/or internal. Amino acid “modification” refers to thealteration of a naturally occurring amino acid to produce anon-naturally occurring amino acid. A “substitution” refers to thereplacement of one or more amino acid residue(s) by another amino acidresidue(s) in the polypeptide. Analogs can contain differentcombinations of alterations including more than one alteration anddifferent types of alterations.

Preferred analogs have one or more amino acid alteration(s) which do notsignificantly affect exendin agonist activity. In regions of the exendinnot necessary for exendin agonist activity, amino acids may be deleted,added or substituted with less risk of affecting activity. In regionsrequired for exendin agonist activity, amino acid alterations are lesspreferred as there is a greater risk of affecting exendin activity. Suchalterations should be conservative alterations. For example, one or moreamino acid residues within the sequence can be substituted by anotheramino acid of a similar polarity which acts as a functional variant.

Conserved regions tend to be more important for protein activity thannon-conserved regions. Known procedures may be used to determine theconserved and non-conserved regions important of receptor activity usingin vitro mutagenesis techniques or deletion analyses and measuringreceptor activity as described by the present disclosure.

Modifications to a specific polypeptide may be deliberate, as throughsite-directed mutagenesis and amino acid substitution during solid-phasesynthesis, or may be accidental such as through mutations in hosts orsystems which produce the polypeptide.

Compounds particularly useful according to the present invention areexendin agonist compounds of the formula (I):

1                  5                       10 Xaa₁ Xaa₂ Xaa₃ Gly ThrXaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈                  15                   20 SerLys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                  25                   30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ LeuLys Asn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Zwherein Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr, Xaa₃ isAsp or Glu; Xaa₄ is Phe, Tyr or naphthylalanine; Xaa₅ is Thr or Ser;Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu, Ile, Val,pentylglycine or Met; Xaa₉ is Leu, Ile, pentylglycine, Val or Met; Xaa₁₀is Phe, Tyr or naphthylalanine; Xaa₁₁ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr,or naphthylalanine; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either SEQ. ID. NOS. 1 or 2. Preferred N-alkyl groups forN-alkylglycine, N-alkylpentylglycine and N-alkylalanine include loweralkyl groups preferably of 1 to about 6 carbon atoms, more preferably of1 to 4 carbon atoms. Suitable compounds include those having amino acidsequences of SEQ. ID. NOS. 5 to 35.

Preferred exendin agonist compounds include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₉ is Leu, pentylglycine or Met.

Preferred compounds include those wherein Xaa₁₃ is Trp or Phe.

Also preferred are compounds where Xaa₄ is Phe or naphthalanine; Xaa₁₁is Ile or Val and Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

According to an especially preferred aspect, Xaa₁₅, Xaa₁₆ and Xaa₁₇ arethe same amino acid reside.

Preferred are compounds wherein Xaa₁₈ is Ser or Tyr, more preferablySer.

Preferably Z is —NH₂.

According to one aspect, preferred are compounds of formula (I) whereinXaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₄ is Phe ornaphthalanine; Xaa₉ is Leu, pentylglycine or Met; Xaa₁₀ is Phe ornaphthalanine; Xaa₁₁ is Ile or Val; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₁₈ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (I) wherein: Xaa₁ is His or Arg; Xaa₂is Gly; Xaa₃ is Asp or Glu; Xaa₄ is Phe or napthylalanine; Xaa₅ is Thror Ser;

Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₆ is Leu or pentylglycine;Xaa₉ is Leu or pentylglycine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ isIle, Val or t-butyltylglycine; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp or Phe;

Xaa₁₄, Xaa₁₅, Xaa₁₆, and Xaa₁₇ are independently Pro, homoproline,thioproline, or N-methylalanine; Xaa₁₈ is Ser or Tyr: and Z is —OH or—NH₂; with the proviso that the compound does not have the formula ofeither SEQ. ID. NOS. 1 or 2. More preferably Z is —NH₂. Especiallypreferred compounds include those having the amino acid sequence of SEQ.ID. NOS. 5, 6, 17, 18, 19, 22, 24, 31, 32 and 35.

According to an especially preferred aspect, provided are compoundswhere Xaa₉ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₁₃ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds are believed to exhibitadvantageous duration of action and to be less subject to oxidativedegration, both in vitro and in vivo, as well as during synthesis of thecompound.

The compounds referenced above form salts with various inorganic andorganic acids and bases. Such salts include salts prepared with organicand inorganic acids, for example, HCl, HBr, H₂SO₄, H₃PO₄,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid,toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonicacid. Salts prepared with bases include ammonium salts, alkali metalsalts, e.g. sodium and potassium salts, and alkali earth salts, e.g.calcium and magnesium salts. Acetate, hydrochloride, andtrifluoroacetate salts are preferred. The salts may be formed byconventional means, as by reacting the free acid or base forms of theproduct with one or more equivalents of the appropriate base or acid ina solvent or medium in which the salt is insoluble, or in a solvent suchas water which is then removed in vacuo or by freeze-drying or byexchanging the ions of an existing salt for another ion on a suitableion exchange resin.

The compounds referenced above form salts with various inorganic andorganic acids and bases. Such salts include salts prepared with organicand inorganic acids, for example, HCl , HBr, H₂SO₄, H₃PO₄,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid,toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonicacid. Salts prepared with bases include ammonium salts, alkali metalsalts, e.g. sodium and potassium salts, and alkali earth salts, e.g.calcium and magnesium salts. Acetate, hydrochloride, andtrifluoroacetate salts are preferred. The salts may be formed byconventional means, as by reacting the free acid or base forms of theproduct with one or more equivalents of the appropriate base or acid ina solvent or medium in which the salt is insoluble, or in a solvent suchas water which is then removed in vacuo or by freeze-drying or byexchanging the ions of an existing salt for another ion on a suitableion exchange resin.

The compounds described above are useful in view of theirpharmacological properties. In particular, the compounds of theinvention possess activity as agents to regulate gastric motility and toslow gastric emptying, as evidenced by the ability to inhibit gastricemptying levels in mammals.

As described in Example 1, gastric emptying was measured in normalSprague Dawley rats using the retention of an acaloric methylcellulosegel containing Phenol Red delivered by gavage. Dye content in stomachsremoved after sacrifice 20 minutes later was determinedspectroscopically, and was compared to that in rats sacrificedimmediately after gavage to assess emptying. The exendins, exendin 3 andexendin 4, dose-dependently inhibited gastric emptying. The ED₅₀ of theresponse to exendin 3 and exendin 4 was 0.1 and 0.08 μg, respectively,demonstrating that the exendins were ˜170-290 times more I potent thanGLP-1 [7-36]NH₂ in inhibiting gastric emptying.

As described in Example 2, the effects of exendin-4 and the exendin-4analogs, exendin-4 acid and ¹⁴Leu,²⁵Phe exendin-4, on inhibition ofgastric emptying were examined. Exendin-4 and the exendin-4 analogs dosedependently inhibiting gastric emptying. The ED₅₀ of exendin-4 was 0.27μg. The ED,s of exendin-4 acid and ¹⁴Leu,²⁵Phe exendin-4 were 0.12 μgand 0.29 μg, respectively, indicating that the potency of the analogswas comparable to that of exendin-4.

As described in Example 3, the effects of exendin-4 and the cloned GLP-1receptor antagonist, exendin [9-39] on gastric emptying were examined.After 20 minutes, the animals treated with exendin-4 showed potentinhibition of gastric emptying, which was not reversed by exendin[9-39]. This occurred regardless of whether the exendin [9-39] wasadministered sc or iv. Exendin [9-39] alone had no effect on gastricemptying.

As noted above, exendin [9-39] is a potent antagonist of GLP-1 whichbinds at the cloned GLP-1 receptor (Fehmann H C, et al., Peptides 15(3):453-6, 1994; Thorens B, et al., Diabetes 42(11): 1678-82, 1993).Surprisingly, however, exendin [9-39] did not block the effect ofexendin-4 on gastric emptying (see FIGS. 4 and 5). These resultsindicate that the effects of exendins and exendin agonists on gastricemptying are not due binding of the exendins at the cloned GLP-1receptor, but instead that the gastric emptying effects of exendins andexendin agonists are due to their action on a separate receptor.

That exendins can act via mechanisms other than those attributable tothe cloned GLP-l receptor is further evidenced by the reported absenceof effect of exendin-4 on inhibition of pentagastrin-induced gastricacid secretion, despite the inhibitory effect of GLP-1 on suchsecretion. Gedulin, et al., Diabetologia, 40(Suppl. 1):A300 (Abstract1181) (1997). Additionally, as described in commonly assigned U.S.Provisional Patent Application Ser. No. 60/034,905, entitled, “Use ofExendins and Agonists Therefor for the Reduction of Food Intake,” filedJan. 7, 1997, peripherally injected exendin inhibited food intake inmice, an action not observed with GLP-1.

Compositions useful in the invention may conveniently be provided in theform of formulations suitable for parenteral (including intravenous,intramuscular and subcutaneous) or nasal or oral administration. In somecases, it will be convenient to provide an exendin or exendin agonistand another anti-emptying agent, such as glucagon, or amylin, or anamylin agonist, in a single composition or solution for administrationtogether. In other cases, it may be more advantageous to administeranother anti-emptying agent separately from said exendin or exendinagonist. A suitable administration format may best be determined by amedical practitioner for each patient individually. Suitablepharmaceutically acceptable carriers and their formulation are describedin standard formulation treatises, e.g., Remington's PharmaceutialSciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A.“Parenteral Formulations of Proteins and Peptides: Stability andStabilizers,” Journal of Parenteral Science and Technology, TechnicalReport No. 10, Supp. 42:2S (1988).

Compounds useful in the invention can be provided as parenteralcompositions for injection or infusion. They can, for example, besuspended in an inert oil, suitably a vegetable oil such as sesame,peanut, olive oil, or other acceptable carrier. Preferably, they aresuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 5.6 to 7.4. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH buffering agents. Useful buffers include forexample, sodium acetate/acetic acid buffers. A form of repository or“depot” slow release preparation may be used so that therapeuticallyeffective amounts of the preparation are delivered into the bloodstreamover many hours or days following transdermal injection or delivery.

The desired isotonicity may be accomplished using sodium chloride orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. Sodium chloride ispreferred particularly for buffers containing sodium ions.

The claimed compositions can also be formulated as pharmaceuticallyacceptable salts (e.g., acid addition salts) and/or complexes thereof.Pharmaceutically acceptable salts are non-toxic salts at theconcentration at which they are administered. The preparation of suchsalts can facilitate the pharmacological use by altering thephysical-chemical characteristics of the composition without preventingthe composition from exerting its physiological effect. Examples ofuseful alterations in physical properties include lowering the meltingpoint to facilitate transmucosal administration and increasing thesolubility to facilitate the administration of higher concentrations ofthe drug.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, phosphate, sulfamate, acetate,citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.Pharmaceutically acceptable salts can be obtained from acids such ashydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, aceticacid, citric acid, lactic acid, tartaric acid, malonic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Suchsalts may be prepared by, for example, reacting the free acid or baseforms of the product with one or more equivalents of the appropriatebase or acid in a solvent or medium in which the salt is insoluble, orin a solvent such as water which is then removed in vacuo or byfreeze-drying or by exchanging the ions of an existing salt for anotherion on a suitable ion exchange resin.

Carriers or excipients can also be used to facilitate administration ofthe compound. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents. The compositions or pharmaceutical composition can beadministered by different routes including intravenously,intraperitoneal, subcutaneous, and intramuscular, orally, topically, ortransmucosally.

If desired, solutions of the above compositions may be thickened with athickening agent such as methyl cellulose. They may be prepared inemulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

Compositions useful in the invention are prepared by mixing theingredients following generally accepted procedures. For example, theselected components may be simply mixed in a blender or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity.

For use by the physician, the compositions will be provided in dosageunit form containing an amount of an exendin or exendin agonist, forexample, exendin 3, exendin 4, with or without another antiemptyingagent. Therapeutically effective amounts of an exendin or exendinagonist for use in the control of gastric emptying and in conditions inwhich gastric emptying is beneficially slowed or regulated are thosethat decrease post-prandial blood glucose levels, preferably to no morethan about 8 or 9 mM or such that blood glucose levels are reduced asdesired. In diabetic or glucose intolerant individuals, plasma glucoselevels are higher than in normal individuals. In such individuals,beneficial reduction or “smoothing” of post-prandial blood glucoselevels, may be obtained. As will be recognized by those in the field, aneffective amount of therapeutic agent will vary with many factorsincluding the age and weight of the patient, the patient's physicalcondition, the blood sugar level or level of inhibition of gastricemptying to be obtained, and other factors.

Such pharmaceutical compositions are useful in causing gastrichypomotility in a subject and may be used as well in other disorderswhere gastric motility is beneficially reduced.

The effective daily anti-emptying dose of the compounds will typicallybe in the range of 0.001 or 0.003 to about 5 mg/day, preferably about0.001 or 0.05 to 2 mg/day and more preferably about 0.001 or 0.01 to 1mg/day, for a 70 kg patient, administered in a single or divided doses.The exact dose to be administered is determined by the attendingclinician and is dependent upon where the particular compound lieswithin the above quoted range, as well as upon the age, weight andcondition of the individual. Administration should begin at the firstsign of symptoms or shortly after diagnosis of diabetes mellitus.Administration may be by injection, preferably subcutaneous orintramuscular. Orally active compounds may be taken orally, howeverdosages should be increased 5-10 fold.

Generally, in treating or preventing elevated, inappropriate, orundesired post-prandial blood glucose levels, the compounds of thisinvention may be administered to patients in need of such treatment in adosage ranges similar to those given above, however, the compounds areadministered more frequently, for example, one, two, or three times aday.

The optimal formulation and mode of administration of compounds of thepresent application to a patient depend on factors known in the art suchas the particular disease or disorder, the desired effect, and the typeof patient. While the compounds will typically be used to treat humanpatients, they may also be used to treat similar or identical diseasesin other vertebrates such as other primates, farm animals such as swine,cattle and poultry, and sports animals and pets such as horses, dogs andcats.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

EXAMPLE 1

The following study was carried out to examine the effects of exendin-3and exendin-4 on gastric emptying and to compare the effects with GLP-1[7-36]NH₂ treatment in rats. This experiment followed a modification ofthe method of Scarpignato, et al., Arch Int. Pharmacodyn. Ther.246:286-94 (1980).

Male Harlan Sprague Dawley (HSD) rats were used. All animals were housedat 22.7±0.8 C in a 12:12 hour light:dark cycle (experiments beingperformed during the light cycle) and were fed and watered ad libitum(Diet LM-485, Teklad, Madison, WIS.). Exendin-3 and exendin-4 weresynthesized according to standard peptide synthesis methods.

The determination of gastric emptying by the method described below wasperformed after a fast of ˜20 hours to ensure that the stomach containedno chyme that would interfere with spectrophotometric absorbancemeasurements.

Conscious rats received by gavage, 1.5 ml of an acaloric gel containing1.5% methyl cellulose (M-0262, Sigma Chemical Co, St Louis, Mo.) and0.05% phenol red indicator. Twenty minutes after gavage, rats wereanesthetized using 5% halothane, the stomach exposed and clamped at thepyloric and lower esophageal sphincters using artery forceps, removedand opened into an alkaline solution which was made up to a fixedvolume. Stomach content was derived from the intensity of the phenol redin the alkaline solution, measured by absorbance at a wavelength of 560nm. In separate experiments on 7 rats, the stomach and small intestinewere both excised and opened into an alkaline solution. The quantity ofphenol red that could be recovered from the upper gastrointestinal tractwithin 20 minutes of gavage was 89±4%; dye which appeared to bindirrecoverably to the gut luminal surface may have accounted for thebalance. To account for a maximal dye recovery of less than 100%,percent of stomach contents remaining after 20 min were expressed as afraction of the gastric contents recovered from control rats sacrificedimmediately after gavage in the same experiment. Percent gastriccontents remaining =(absorbance at 20 min)/(absorbance at 0 mm)×100.

In baseline studies, with no drug treatment, gastric emptying over 20min was determined. In dose-response studies, rats were treated with 0,0.01, 0.1, 0.3, 1, 5, 10, or 100 μg of exendin 3, exendin 4, orGLP-1(7-36)NH₂. The results are shown in FIG. 2. FIG. 2 shows thatexendins 3 and 4 inhibited gastric emptying with approximately the sameED₅₀ of 0.1 μg, whereas GLP-1(7-36)NH₂ has an ED₅₀ of approximately 9μg, indicating that the exendins are ˜90 fold more potent than GLP-1 atinhibiting gastric emptying.

As shown in Table I, exendin-3 and exendin-4 were found to be potentinhibitors of gastric emptying. The effect of rat amylin on gastricemptying is also provided as a second positive control and forcomparitive purposes.

TABLE I GLP-1 (7-36) NH₂ Exendin-3 Exendin-4 Rat Amylin DOSE μg %remaining *(n) SEM % remaining *(n) SEM % remaining *(n) SEM % remaining*(n) SEM Saline 48.00 (16) 3.50 46.760 (15) 2.360 46.000 (17) 2.00048.00 (17) 3.5 Control 0.010 no data 58.240 (3) 3.180 no data 2.00037.60 (2) 2.50 0.100 42.00 (7) 6.50 70.770 (3) 5.600 72.000 (3) 12.00052.70 (6) 6.30 0.300 29.60 (7) 3.50 86.420 (3) 6.160 98.000 (2) 4.00088.20 (4) 3.00 1.000 37.20 (9) 2.70 95.330 (3) 0.790 105.000 (1) 0.00096.80 (9) 2.80 3.000 56.60 (10) 6.10 108.00 (4) 2.70 10.000 87.90 (11)2.70 101.760 (3) 3.390 112.000 (3) 2.000 101.10 (6) 3.60 100.000 103.60(7) 2.80 103.640 (3) 2.260 103.000 (3) 3.000 101.20 (2) 2.80 *percent ofgastric contents remaining 20 minutes after gavage.

EXAMPLE 2

The effects of exendin-4 analogs on inhibition of gastric emptying wereexamined, and compared to the effects of exendin-4, according to themethods described in Example 1. Male HSD rats were treated with 0.01,0.1, 0.3, 1, 10 and 100 μg of exendin-4 0.01, 0.03, 0.1, 1, 10 and 100μg exendin-4 acid, [SEQ. ID. NO. 36] and 0.1, 0.3, 1, 10 and 100 μg of¹⁴Leu,²⁵Phe exendin-4 [SEQ. ID. NO. 5]. Exendin-3, exendin-4 acid and¹⁴Leu,²⁵Phe were synthesized according to standard peptide synthesismethods. The results, shown in FIG. 3 and Table II, show that theexendin agonists, exendin-4 acid and ¹⁴Leu,²⁵Phe exendin-4, are potentinhibitors of gastric emptying. The EC₅₀ of exendin-4 was 0.27 μg. TheEC₅₀ s of exendin-4 acid and ¹⁴Leu,²⁵Phe exendin-4 were comparable (0.12μg and 0.29 μg, respectively).

TABLE II Compound EC₅₀ (μg) exendin-4 0.27 exendin-4 acid 0.12 ¹⁴Leu,²⁵Phe exendin-4 0.29

The ability of exendin [9-39], an antagonist of exendin's effects at thecloned GLP-1 receptor, to antagonize the gastric emptying inhibitioneffect of exendin-4 and GLP-1 [7-36]NH₂ was examined according to themethods described in Example 1. Rats were treated with 1.0 μg exendin-4,1.0 μg exendin-4 with 0.3 mg exendin [9-39], 10 μg GLP-1 [7-36]NH₂ 10 μgGLP-1 [7-36]NH₂ with 0.3 mg exendin [9-39] or with 0.3 mg exendin 9-39alone. In these studies, exendin [9-39] was give both subcutaneously(sc) and intravenously (iv). The results of these experiments are shownin FIGS. 4-7.

As shown in FIGS. 4 and 5, after 20 minutes, the animals treated withexendin-4 showed extremely potent inhibition of gastric emptying, whichwas not reversed by exendin [9-39]. This occurred regardless of whetherthe exendin [9-39] was administered sc or iv. Exendin [9-39] alone hadno effect on gastric emptying.

As discussed above, exendin [9-39] is a potent antagonist of GLP-1 whichbinds at the cloned GLP-1 receptor (Fehmann H C, et al., Peptides 15(3):453-6, 1994; Thorens B. et al., Diabetes 42(11): 1678-82, 1993).Surprisingly, however, exendin [9-39] did not block the effect ofexendin-4 on gastric emptying (see FIGS. 4 and 5). These resultsindicate that the effects of exendins on gastric emptying are not duebinding of the exendins at the cloned GLP-1 receptor, but instead thatthe gastric emptying effects of exendins are due to a differentreceptor.

That exendin [9-39] did not block the effect of GLP-1 [7-36]NH₂ ongastric emptying (see FIGS. 6 and 7) indicates that, in its effects ongastric emptying, GLP-1 is also acting at a receptor other than thecloned CLP-1 receptor (at which exendin [9-39] is a potent antagonist).

EXAMPLE 4 Preparation of Amidated Peptide Having SEQ. ID. No. [5]

The above-identified peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.). In general, single-coupling cycles were usedthroughout the synthesis and Fast Moc (HBTU activation) chemistry wasemployed. However, at some positions coupling was less efficient thanexpected and double couplings were required. In particular, residuesAsp₉, Thr₇ and Phe₆ all required double coupling. Deprotection (Fmocgroup removal)of the growing peptide chain using piperidine was notalways efficient. Double deprotection was required at positions Arg₂₀,Val₁₉ and Leu₁₄. Final deprotection of the completed peptide resin wasachieved using a mixture of triethylsilane (0.2 mL), ethanedithiol (0.2mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid (15 mL)according to standard methods (Introduction to Cleavage Techniques,Applied Biosystems, Inc.) The peptide was precipitated in ether/water(50 mL) and centrifuged. The precipitate was reconstituted in glacialacetic acid and lyophilized. The lyophilized peptide was dissolved inwater). Crude purity was about 55%.

Used in purification steps and analysis were Solvent A (0.1% TFA inwater) and Solvent B (0.1% TFA in ACN).

The solution containing peptide was applied to a preparative C-18 columnand purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purityof fractions was determined isocratically using a C-18 analyticalcolumn. Pure fractions were pooled furnishing the above-identifiedpeptide. Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide gave product peptide havingan observed retention time of 14.5 minutes. Electrospray MassSpectrometry (M): calculated 4131.7; found 4129.3.

EXAMPLE 5 Preparation of Peptide Having SEQ. ID. NO. [6]

The above-identified peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis were Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 25% to 75% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide gave product peptide having an observed retentiontime of 21.5 minutes. Electrospray Mass Spectrometry (M): calculated4168.6; found 4171.2.

EXAMPLE 6 Preparation of Peptide Having SEQ. ID. NO. [7]

The above-identified peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis were Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide gave product peptide having an observed retentiontime of 17.9 minutes. Electrospray Mass Spectrometry (M): calculated4147.6; found 4150.2.

EXAMPLE 7 Preparation of Petide Having SEQ. ID. NO. [8]

The above-identified peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis were Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 35% to 65% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide gave product peptide having an observed retentiontime of 19.7 minutes. Electrospray Mass Spectrometry (M): calculated4212.6; found 4213.2.

EXAMPLE 8 Preparation of Peptide Having SEQ. ID. NO. [9]

The above-identified peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis were Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 50% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide gave product peptide having an observed retentiontime of 16.3 minutes. Electrospray Mass Spectrometry (M): calculated4262.7; found 4262.4.

EXAMPLE 9 Preparation of Peptide Having SEQ. ID. NO. [10]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4172.6

EXAMPLE 10 Preparation of Peptide Having SEQ. ID. NO. [11]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4224.7.

EXAMPLE 11 Preparation of Peptide Having SEQ. ID. NO. [12]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4172.6

EXAMPLE 12 Preparation of Peptide Having SEQ. ID. NO. [13]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4186.6

EXAMPLE 13 Preparation of Peptide Having SEQ. ID. NO. [14]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4200.7

EXAMPLE 14 Preparation of Peptide Having SEQ. ID. NO. [15]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4200.7

EXAMPLE 15 Preparation of Peptide Having SEQ. ID. NO. [16]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4202.7.

EXAMPLE 16 Preparation of Peptide Having SEQ. ID. NO. [17]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4145.6.

EXAMPLE 17 Preparation of Peptide Having SEQ. ID. NO. [18]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4184.6.

EXAMPLE 18 Preparation of Peptide Having SEQ. ID. NO. [19]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4145.6.

EXAMPLE 19 Preparation of Peptide Having SEQ. ID. NO. [20]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4224.7.

EXAMPLE 20 Preparation of Peptide Having SEQ. ID. NO. [21]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4172.6.

EXAMPLE 21 Preparation of Peptide Having SEQ. ID. NO. [22]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4115.5.

EXAMPLE 22 Preparation of Peptide Having SEQ. ID. NO. [23]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 4. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4188.6.

EXAMPLE 23 Preparation of Peptide Having SEQ. ID. NO. [24]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBRA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4131.6.

EXAMPLE 24 Preparation of Peptide Having SEQ. ID. NO. [25]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4172.6.

EXAMPLE 27 Preparation of Peptide Having SEQ. ID. No. [28]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the thioproline positions 38, 37 and 36. Used in analysisare Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30minutes) of the lyophilized peptide is then carried out to determine theretention time of the product peptide. Electrospray Mass Spectrometry(M): calculated 4246.8.

EXAMPLE 28 Preparation of Peptide Having SEQ. ID. NO. [29]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the homoproline positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide.

EXAMPLE 25 Preparation of Peptide Having SEQ. ID. NO. [26]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Used in analysis are Solvent A(0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of thelyophilized peptide is then carried out to determine the retention timeof the product peptide. Electrospray Mass Spectrometry (M): calculated4145.6.

EXAMPLE 26 Preparation of Peptide Having SEQ. ID. NO. [27]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the thioproline positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 4266.8.

Electrospray Mass Spectrometry (M): calculated 4250.8.

EXAMPLE 29 Preparation of Peptide Having SEQ. ID. NO. [30]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the homoproline positions 38, 37, and 36. Used in analysisare Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30minutes) of the lyophilized peptide is then carried out to determine theretention time of the product peptide. Electrospray Mass Spectrometry(M): calculated 4234.8.

EXAMPLE 30 Preparation of Peptide Having SEQ. ID. NO. [31]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the thioproline positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 4209.8.

EXAMPLE 31 Preparation of Peptide Having SEQ. ID. NO. [32]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the homoproline positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 4193.7.

EXAMPLE 32 Preparation of Peptide Having SEQ. ID. NO. [33]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g). using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the N-methylalanine positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 3858.2.

EXAMPLE 33 Preparation of Peptide Having SEQ. ID. NO. [34]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the N-methylalanine positions 38, 37 and 36. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 3940.3.

EXAMPLE 34 Preparation of Peptide Having SEQ. ID. NO. [35]

The above-identified peptide is assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Example 1. Additional double couplings arerequired at the N-methylalanine positions 38, 37, 36 and 31. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry (M): calculated 3801.1.

EXAMPLE 35 Preparation of C-terminal Carboxylin Acid PeptidesCorresponding to the above C-terminal Amide Sequences

The above peptides are assembled on the so called Wang resin(p-alkoxybenzylalacohol resin (Bachem, 0.54 mmole/g)) usingFmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from theresin, deprotected and purified in a similar way to Example 1. Used inanalysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide is then carried out todetermine the retention time of the product peptide. Electrospray MassSpectrometry provides an experimentally determined (M).

1. A method of reducing gastric motility in a subject in need thereofcomprising administering to said subject an amount of an exendineffective for reducing gastric motility.
 2. A method of delaying gastricemptying in a subject in need thereof comprising administering to saidsubject an amount of an exendin effective for delaying gastric emptying.3. The method according to claim 1 or 2 wherein said exendin isexendin-3.
 4. The method according to claim 1 or 2 wherein said exendinis exendin-4.
 5. The method according to claim 1 or 2 wherein saidsubject is undergoing a gastrointestinal diagnostic procedure.
 6. Themethod according to claim 5 wherein said gastrointestinal diagnosticprocedure is a radiological examination.
 7. The method according toclaim 6 wherein said gastric gastrointestinal diagnostic procedure ismagnetic resonance imaging.
 8. The method according to claim 1 or 2wherein said subject is suffering from a gastrointestinal disorder.
 9. Amethod of reducing gastric motility in a subject in need thereofcomprising administering to said subject an amount of an exendin analogeffective for reducing gastric motility, wherein said exendin analog isselected from a peptide compound of the formula: 1                  5                      10 Xaa₁ Xaa₂ Xaa₃ Gly ThrXaa₄ Xaa₅, Xaa₆ Xaa₇ Xaa₈                   15                 20 SerLys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                  25                   30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ LeuLys Asn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Z

wherein: Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ isAsp or Glu; Xaa₄ is Phe, Tyr or naphthylalanine; Xaa₅ is Thr or Ser;Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu, Ile, Val,pentylglycine or Met; Xaa₉ is Leu, Ile, pentylglycine, Val or Met; Xaa₁₀is Phe, Tyr or naphthylalanine; Xaa₁₁ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr,or naphthylalanine; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either exendin-3 or exendin-4 and pharmaceutically acceptablesalts thereof.
 10. A method of reducing gastric motility in a subject inneed thereof comprising administering to said subject an amount of anexendin analog effective for reducing gastric motility, wherein saidexendin analog is selected from a peptide compound of the formula: 1                 5                       10 Xaa₁ Xaa₂ Xaa₃ Gly ThrXaa₄ Xaa₅, Xaa₆ Xaa₇ Xaa₈                  15                  20 SerLys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                 25                    30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ LeuLys Asn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Z

wherein: Xaa₁ is His or Arg; Xaa₂ is Ser or Gly; Xaa₃ is Asp or Glu;Xaa₄ is Phe or naphthylalanine; Xaa₅ is Thr or Ser; Xaa₆ is Ser or Thr;Xaa₇ is Asp or Glu; Xaa₈ is Leu or pentylglycine Xaa₉ is Leu orpentylglycine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile, Val ortert-butylglycine; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp or Phe; Xaa₁₄,Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro, homoprolineor N-methylalanine; Xaa₁₈ is Ser or Tyr; and Z is —OH or —NH₂; with theproviso that the compound does not have the formula of either exendin-3or exendin-4 and pharmaceutically acceptable salts thereof.
 11. A methodof delaying gastric emptying in a subject in need thereof comprisingadministering to said subject an amount of an exendin analog effectivefor delaying gastric emptying, wherein said exendin analog is selectedfrom a peptide compound of the formula: 1                 5                       10 Xaa₁ Xaa₂ Xaa₃ Gly ThrXaa₄ Xaa₅, Xaa₆ Xaa₇ Xaa₈                  15                  20 SerLys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                 25                    30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ LeuLys Asn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Z

wherein: Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ isAsp or Glu; Xaa₄ is Phe, Tyr or naphthylalanine; Xaa₅ is Thr or Ser;Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu, Ile, Val,pentylglycine or Met; Xaa₉ is Leu, Ile, pentylglycine, Val or Met; Xaa₁₀is Phe, Tyr or naphthylalanine; Xaa₁₁ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr,or naphthylalanine; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either exendin-3 or exendin-4 and pharmaceutically acceptablesalts thereof.
 12. A method of delaying gastric emptying in a subject inneed thereof comprising administering to said subject an amount of anexendin analog effective for delaying gastric emptying, wherein saidexendin analog is selected from a peptide compound of the formula: 1                 5                       10 Xaa₁ Xaa₂ Xaa₃ Gly ThrXaa₄ Xaa₅, Xaa₆ Xaa₇ Xaa₈                  15                  20 SerLys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                 25                    30 Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ LeuLys Asn Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈-Z

wherein: Xaa₁ is His or Arg; Xaa₂ is Ser or Gly; Xaa₃ is Asp or Glu;Xaa₄ is Phe or naphthylalanine; Xaa₅ is Thr or Ser; Xaa₆ is Ser or Thr;Xaa₇ is Asp or Glu; Xaa₈ is Leu or pentylglycine Xaa₉ is Leu orpentylglycine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile, Val ortert-butylglycine; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp or Phe; Xaa₁₄,Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro, homoprolineor N-methylalanine; Xaa₁₈ is Ser or Tyr; and Z is —OH or —NH₂; with theproviso that the compound does not have the formula of either exendin-3or exendin-4 and pharmaceutically acceptable salts thereof.
 13. Themethod according to claim 9, 10, 11, or 12 wherein said subject isundergoing a gastrointestinal diagnostic procedure.
 14. The methodaccording to claim 13 wherein said gastrointestinal diagnostic procedureis a radiological examination.
 15. The method according to claim 14wherein said gastric gastrointestinal diagnostic procedure is magneticresonance imaging.
 16. The method according to claim 9, 10, 11, or 12wherein said subject is suffering from a gastrointestinal disorder. 17.A method of reducing gastric motility in a subject in need thereofcomprising administering to said subject an amount of exendin-4effective for reducing gastric motility.
 18. A method of delayinggastric emptying in a subject in need thereof comprising administeringto said subject an amount of exendin-4 effective for delaying gastricemptying.
 19. The method according to claim 17 or 18 wherein saidsubject is undergoing a gastrointestinal diagnostic procedure.
 20. Themethod according to claim 19 wherein said gastrointestinal diagnosticprocedure is a radiological examination.
 21. The method according toclaim 20 wherein said gastric gastrointestinal diagnostic procedure ismagnetic resonance imaging.
 22. The method according to claim 17 or 18wherein said subject is suffering from a gastrointestinal disorder.